An in vivo screening system and screening method

Abstract

The application discloses an in vivo screening system and a screening method. The system comprises a loading module for taking fish from a fish tank and pushing the fish to move in a main hose; the drug release imaging module comprises a microfluidic chip, the microfluidic chip comprises a main channel and a plurality of channels connected with the main channel; the fish loading channel is connected with an output channel of the loading module to receive the fish of the output channel; the gas inlet channel is used for injecting gas to divide the liquid bubble loaded with the fish and push the liquid bubble to move in the channel through the injected gas; the drug release imaging module is used for injecting a drug solution into the liquid bubble through a drug release channel; and whether the fish needs to be pushed into a second observation channel after entering the first observation channel is determined according to the orientation of the fish in the liquid bubble, so that the fish is observed. The embodiment of the application is favorable for improving the efficiency and accuracy of in vivo screening experiments and saving experimental cost. The application can be widely applied in the field of automatic biological experiment technology.

Description

Technical Field

[0001] This invention relates to the field of automated biological experimental technology, and in particular to an in vivo screening system and screening method. Background Technology

[0002] In drug development, biological simulation experiments are often required. In some embodiments, zebrafish share approximately 70% of their genes with humans and possess easily manipulated fluorescent labeling characteristics, making them a strong contender for applications in drug discovery and screening. One approach uses agarose to immobilize zebrafish, enabling fluorescent imaging of the zebrafish brain. While the immobilization effect is significant, the animals are encapsulated in agar, hindering behavioral assessment. Another approach utilizes microfluidics for drug testing with zebrafish. However, this method lacks automated movement and immobilization capabilities, is complex, inefficient, and requires continuous perfusion for drug delivery, resulting in high drug consumption and potentially excessive economic costs. Summary of the Invention

[0003] The purpose of this invention is to at least partially solve one of the technical problems existing in the prior art.

[0004] Therefore, the purpose of this invention is to provide an in vivo screening system and screening method.

[0005] To achieve the above-mentioned technical objectives, the technical solutions adopted in the embodiments of the present invention include the following aspects:

[0006] On one hand, embodiments of the present invention provide an in vivo screening system, including: a loading module and a drug release imaging module; wherein, the loading module is used to remove fish from a fishpond and push the fish to move in a main flexible tube; the drug release imaging module includes a microfluidic chip, the microfluidic chip including a main channel and a plurality of channels connecting the main channel, the plurality of channels including a gas inlet channel, a fish loading channel, a liquid outlet channel, a drug release channel, a first observation channel, a second observation channel, and a fish release channel arranged sequentially along the movement direction of the fish; the fish loading channel is connected to the output channel of the loading module to receive fish from the output channel; the gas inlet channel is used to inject gas to divide the vacuoles containing the fish and push the vacuoles to move in the channel by the injected gas; the drug release imaging module is used to: inject a drug solution into the vacuoles through the drug release channels; and determine whether the fish needs to be pushed into the second observation channel after entering the first observation channel, based on the orientation of the fish in the vacuoles, in order to observe the fish. This application embodiment achieves full automation and improves experimental efficiency by designing various channels in a microfluidic chip and controlling the movement of fish along these channels. The fish is loaded into vacuoles, and the vacuoles are divided by a gas inlet channel to allow drug release at the connection between the drug release channel and the main channel, improving experimental reliability. Furthermore, by fixing the fish within tiny vacuoles, this embodiment requires only a very small amount of drug to complete the experiment, significantly saving on experimental drugs and costs. This application embodiment is beneficial for improving the efficiency and accuracy of in vivo screening experiments and saving experimental costs.

[0007] In addition, the in vivo screening system according to the above embodiments of the present invention may also have the following additional technical features:

[0008] Further, in the in vivo screening system of this embodiment, the loading module includes: a first pump for injecting air bubbles into the bottom of the fishpond to allow fish to enter the main hose inserted into the fishpond; a second pump connected to the main hose via the first hose; and a third pump connected to the main hose via the second hose. A first switch is disposed on the main hose near the fishpond, a second switch is disposed on the first hose, a third switch is disposed on the second hose, and a fourth switch is disposed on the main hose near the drug release imaging module. A first image acquisition device is used to acquire a first image of a first region, the first region being the main hose region between the first hose and the second hose. The loading module is used to: turn on the first pump and the third pump, and turn on the first switch and the third switch to draw the fish into the main hose and push the fish to move within the main hose; if the first image contains a fish, turn on the second pump, and turn on the second switch and the fourth switch to push the fish to the drug release imaging module.

[0009] Further, in one embodiment of the present invention, the drug release imaging module includes: a fourth pump connected to the gas inlet channel via a fifth switch, a sixth switch connected to the liquid outlet channel, a fifth pump connected to the drug release channel, a seventh pump connected to the second observation channel via a seventh switch, a sixth pump connected to the first observation channel via an eighth switch, and a ninth switch connected to the fish release channel; a second image acquisition device for acquiring images of each channel of the microfluidic chip, wherein the second region is the main channel region between the fish loading channel and the liquid outlet channel; the drug release imaging module is used to: if the second region contains the fish and the orientation is the same as a preset orientation, turn on the fourth pump and the sixth pump, and turn on the fifth switch and the fifth switch. Eight switches are used to divide the bubble; the fifth pump is turned on to inject a drug solution into the bubble; once the fish moves to the first observation channel, the fish is observed and analyzed by an image processing device to complete the experiment; or, if the second area contains the fish and its orientation is different from the preset orientation, the fourth and sixth pumps are turned on, and the fifth and eighth switches are turned on to divide the bubble; the fifth pump is turned on to inject a drug solution into the bubble; once the fish moves to the first observation channel, the sixth pump is controlled to push forward, the seventh pump to retract backward, and the seventh and eighth switches are turned on to move the fish from the first observation channel to the second observation channel; the fish is observed and analyzed by an image processing device to complete the experiment.

[0010] Furthermore, in one embodiment of the present invention, the cross-sectional area of ​​the first observation channel near the main channel is a first area, and the cross-sectional area of ​​the first observation channel away from the main channel is a second area, wherein the first area is larger than the second area; the portion of the first observation channel away from the main channel is flat.

[0011] Furthermore, in one embodiment of the present invention, the fish pond is a funnel-shaped container that is wider at the top and narrower at the bottom.

[0012] On the other hand, embodiments of the present invention propose an in vivo screening method applied to the aforementioned in vivo screening system, the method comprising:

[0013] Remove the fish from the fish tank and move it through the main hose;

[0014] If the fish enters the drug release imaging module, gas is injected to divide the vacuoles containing the fish and to propel the vacuoles in the main channel by the injected gas.

[0015] If the fish reaches the connection between the drug release channel and the main channel, the drug solution is injected into the vacuoles;

[0016] Based on the orientation of the fish in the bubble, determine whether the fish needs to be pushed into the second observation channel after entering the first observation channel in order to observe the fish.

[0017] Further, in the in vivo screening method of this embodiment of the invention, a first pump is connected to the bottom of the fishpond, a second pump is connected to the main hose via a first flexible tube, and a third pump is connected to the main hose via a second flexible tube; a first switch is provided on the main hose near the fishpond, a second switch is provided on the first hose, a third switch is provided on the second hose, and a fourth switch is provided on the main hose near the drug release imaging module; a first image acquisition device is used to acquire a first image of a first region, the first region being the main hose region between the first hose and the second hose; the method further includes:

[0018] Turn on the first pump and the third pump, turn on the first switch and the third switch to draw the fish into the main hose and push the fish to move in the main hose;

[0019] If the first image contains a fish, turn on the second pump, the second switch, and the fourth switch to push the fish to the drug release imaging module.

[0020] Further, in the in vivo screening method of this embodiment, the drug release imaging module includes: a fourth pump connected to the gas inlet channel via a fifth switch, a sixth switch connected to the liquid outlet channel, a fifth pump connected to the drug release channel, a seventh pump connected to the second observation channel via a seventh switch, a sixth pump connected to the first observation channel via an eighth switch, and a ninth switch connected to the fish release channel; a second image acquisition device for acquiring images of each channel of the microfluidic chip, wherein the second region is the main channel region between the fish loading channel and the liquid outlet channel, and the method further includes:

[0021] If the second area contains the fish and its orientation is the same as the preset orientation, turn on the fourth and sixth pumps, and turn on the fifth and eighth switches to divide the vacuoles; turn on the fifth pump to inject the drug solution into the vacuoles; wait for the fish to move to the first observation channel, and observe and analyze the fish through the image processing device to complete the experiment;

[0022] or,

[0023] If the second region contains the fish and its orientation differs from the preset orientation, the fourth and sixth pumps are activated, as are the fifth and eighth switches, to separate the vacuoles; the fifth pump is activated to inject the drug solution into the vacuoles; once the fish moves to the first observation channel, the sixth pump is pushed forward, the seventh pump is retracted, and the seventh and eighth switches are activated to move the fish from the first observation channel to the second observation channel; the fish is observed and analyzed using an image processing device to complete the experiment.

[0024] On the other hand, embodiments of the present invention provide an in vivo screening device, comprising:

[0025] At least one processor;

[0026] At least one memory for storing at least one program;

[0027] When the at least one program is executed by the at least one processor, the at least one processor implements the in vivo screening method described above.

[0028] On the other hand, embodiments of the present invention provide a storage medium storing a processor-executable program, which, when executed by a processor, is used to implement the above-described in vivo screening method.

[0029] The system provided in this embodiment of the invention includes: a loading module and a drug release imaging module; wherein, the loading module is used to remove fish from a fishpond and push the fish to move in a main flexible tube; the drug release imaging module includes a microfluidic chip, the microfluidic chip includes a main channel and several channels connecting the main channel, the several channels including a gas inlet channel, a fish loading channel, a liquid outlet channel, a drug release channel, a first observation channel, a second observation channel, and a fish release channel arranged sequentially along the movement direction of the fish; the fish loading channel is connected to the output channel of the loading module to receive the fish from the output channel; the gas inlet channel is used to inject gas to divide the bubble containing the fish, and to push the bubble to move in the channel by the injected gas; the drug release imaging module is used to: inject a drug solution into the bubble through the drug release channel; and determine whether the fish needs to be pushed into the second observation channel after entering the first observation channel, based on the orientation of the fish in the bubble, in order to observe the fish. This application embodiment achieves full automation and improves experimental efficiency by designing various channels in a microfluidic chip and controlling the movement of fish along these channels. The fish is loaded into vacuoles, and the vacuoles are divided by a gas inlet channel to allow drug release at the connection between the drug release channel and the main channel, improving experimental reliability. Furthermore, by fixing the fish within tiny vacuoles, this embodiment requires only a very small amount of drug to complete the experiment, significantly saving on experimental drugs and costs. This application embodiment is beneficial for improving the efficiency and accuracy of in vivo screening experiments and saving experimental costs. Attached Figure Description

[0030] To more clearly illustrate the technical solutions in the embodiments of the present invention or the prior art, the following description is provided with accompanying drawings of the relevant technical solutions in the embodiments of the present invention or the prior art. It should be understood that the accompanying drawings described below are only for the purpose of clearly illustrating some embodiments of the technical solutions of the present invention. For those skilled in the art, other drawings can be obtained based on these drawings without any creative effort.

[0031] Figure 1 A schematic diagram of the structure of an embodiment of the in vivo screening system provided by the present invention;

[0032] Figure 2 A schematic diagram illustrating the fish-absorbing principle of one embodiment of the fishpond structure provided by the present invention;

[0033] Figure 3 A schematic diagram of the structure of an embodiment of the microfluidic chip provided by the present invention;

[0034] Figure 4 A schematic diagram of the structure of an embodiment of the observation channel provided by the present invention;

[0035] Figure 5(a) is a schematic diagram of multi-organ imaging of an embodiment of the transgenic animal provided by the present invention;

[0036] Figure 5(b) is a schematic diagram of multi-organ imaging of another embodiment of the transgenic animal provided by the present invention;

[0037] Figure 5(c) is a schematic diagram of multi-organ imaging of another embodiment of the transgenic animal provided by the present invention;

[0038] Figure 6 A schematic flowchart of one embodiment of the fish orientation determination process provided by the present invention;

[0039] Figure 7 A flowchart illustrating one embodiment of the experimental process provided by the present invention;

[0040] Figure 8 A schematic flowchart illustrating another embodiment of the experimental process provided by the present invention;

[0041] Figure 9(a) is a schematic flowchart of an embodiment of the drug release process provided by the present invention;

[0042] Figure 9(b) is a schematic diagram of the results of an embodiment of the determination of the molar absorbance of methylene blue provided by the present invention;

[0043] Figure 9(c) is a schematic diagram of the results of an embodiment of the concentration comparison in the experiment shown in Figure 9(a) provided by the present invention;

[0044] Figure 9(d) is a schematic diagram of the results of an embodiment of the absorbance comparison in the experiment shown in Figure 9(a) provided by the present invention;

[0045] Figure 9(e) is a schematic diagram of the results of an embodiment of the absorption spectrum in the experiment shown in Figure 9(a) provided by the present invention;

[0046] Figure 9(f) is a schematic diagram of the results of one embodiment of the concentration gradient in the experiment shown in Figure 9(a) provided by the present invention;

[0047] Figure 10 A schematic diagram illustrating the time consumption of each step in one embodiment of the in vivo screening process provided by the present invention;

[0048] Figure 11(a) is a schematic diagram of the results of an embodiment of the health assessment of fish after in vivo screening provided by the present invention;

[0049] Figure 11(b) is a schematic diagram of the results of another embodiment of the health assessment of fish after in vivo screening provided by the present invention;

[0050] Figure 12(a) is a schematic diagram of the results of an embodiment of larval imaging through the observation channel provided by the present invention;

[0051] Figure 12(b) is a schematic diagram of the results of an embodiment of heart rate measurement based on ventricular ROI provided by the present invention;

[0052] Figure 12(c) is a schematic diagram of the results of an embodiment of the present invention based on fitting analysis of the ventricular wall motion in the cardiac cycle;

[0053] Figure 12(d) is a schematic diagram of the results of an embodiment of the wild zebrafish heart cycle sequence provided by the present invention;

[0054] Figure 12(e) is a schematic diagram of the results of an embodiment of the transgenic zebrafish heart cycle sequence provided by the present invention;

[0055] Figure 12(f) is a schematic diagram of the results of one embodiment of the gray value changes in the center of the ventricle of zebrafish provided by the present invention. Detailed Implementation

[0056] The embodiments of the present invention are described in detail below. Examples of these embodiments are shown in the accompanying drawings, wherein the same or similar reference numerals denote the same or similar elements or elements having the same or similar functions throughout. The embodiments described below with reference to the accompanying drawings are exemplary and are only used to explain the present invention, and should not be construed as limiting the present invention. The step numbers in the following embodiments are set only for ease of explanation, and there is no limitation on the order between the steps. The execution order of each step in the embodiments can be adaptively adjusted according to the understanding of those skilled in the art.

[0057] In drug development, cardiotoxicity is a common cause of failure. Current strategies for screening candidate drugs for adverse cardiac symptoms involve in vitro cardiomyocyte analysis and the use of animal models. While the former allows for rapid screening of cardiotoxicity characteristics, it cannot reflect the effects on the entire organism. Conversely, animal model-based methods can characterize cardiotoxicity in more detail, but their widespread use is limited by their high cost and time consumption.

[0058] Zebrafish (Danio rerio), as a unique model organism, share approximately 70% of its genes with humans. Its highly transparent body organs, rapid development cycle, and easily manipulated fluorescent markers make it a promising candidate for drug discovery and screening. Zebrafish eggs begin circulating in the bloodstream just 24 hours after fertilization (hpf). Compared to mammals, the single-heart, single-ventricle structure of zebrafish provides a simpler and smaller model. However, their ventricle-to-atrium ratio and heart rate are comparable to those of mice and humans, respectively. These characteristics also make zebrafish an important research tool in the detection of cardiovascular toxicity. However, traditional zebrafish experiments still largely rely on manual manipulation, which is not only inefficient but also susceptible to operator variability, potentially leading to instability and unreliability of experimental results.

[0059] With the development of automation control technology, more and more research is emerging on automated systems that use zebrafish as model organisms for drug screening. One embodiment uses a single system to image zebrafish in any direction at cellular resolution within a single channel; however, this platform system is complex, costly, and technically demanding. Another embodiment uses a non-contact method, employing an acousto-fluidic rotating tweezers platform to manipulate and observe juvenile zebrafish. However, this system requires anesthesia of the juveniles, which can cause neurological damage to the immature zebrafish brain, reduce heart rate, and lead to high mortality. Especially during prolonged exposure, it can interfere with the test drug, affecting the assessment of actual toxic side effects. Currently, agarose is also used to immobilize zebrafish, enabling fluorescence imaging of the zebrafish brain. While the immobilization effect is significant, the animals in this platform are encapsulated in agar, prohibiting behavioral evaluation. Microfluidic technology has also been used in recent years to arrange and immobilize zebrafish, thereby achieving stable observation of them. One embodiment uses microfluidic technology to immobilize a single zebrafish for drug testing, successfully recording brain fluorescence under drug influence. However, this method is complex and cannot automate the movement and immobilization of the zebrafish, resulting in low efficiency. Another embodiment uses a microfluidic channel to immobilize multiple juvenile fish. After collecting brain information, deep learning is used to further advance high-throughput drug screening based on zebrafish. However, continuous drug administration is required during the injection phase to fix the zebrafish's position, thus preventing the screening that consumes only trace amounts of drug.

[0060] To address this issue, this application proposes a fully automated zebrafish loading and observation system. This system, combining microfluidics and automatic control technologies, achieves automated loading, automated drug administration, continuous observation, and data acquisition of zebrafish, enabling the testing of bright-field and fluorescence microscopic imaging. The system can be used for imaging at various resolutions. This application constructs a fully automated experimental process from zebrafish loading to data acquisition, providing a more stable and reliable experimental platform. Specifically, through computer vision feedback, this application automatically loads zebrafish into vacuoles of constant volume. The system controls the position of the vacuoles within a microfluidic chip to move the zebrafish. The system controls the amount of drug injected into the vacuoles within the chip, thereby configuring drug solutions of different concentrations. Different oriented slots / channels are designed within the chip to accommodate zebrafish with different orientations, enabling zebrafish fixation and post-drug treatment data observation. This method eliminates the need for gel fixation and anesthesia of the zebrafish, and the entire process is automated. The system in this application embodiment can reduce the complexity of fixed zebrafish and multi-concentration gradient drug detection. Through a fully automated detection process, it reduces human interference, accelerates the screening of drug toxicity and side effects, and thus further simplifies the drug screening process.

[0061] The in vivo screening system and implementation method according to embodiments of the present invention are described in detail below with reference to the accompanying drawings. First, an in vivo screening system according to an embodiment of the present invention is introduced, the system specifically including:

[0062] Loading module, drug release imaging module;

[0063] The loading module is used to remove fish from the fish tank and push the fish through the main hose.

[0064] The drug release imaging module includes a microfluidic chip, which includes a main channel and several channels connecting the main channel. The several channels include a gas inlet channel, a fish loading channel, a liquid outlet channel, a drug release channel, a first observation channel, a second observation channel, and a fish release channel arranged sequentially along the direction of fish movement.

[0065] The fish loading channel is connected to the output channel of the loading module to receive fish from the output channel; the gas inlet channel is used to inject gas to divide the bubble of the loaded fish and to propel the bubble within the channel by the injected gas; the drug release imaging module is used for:

[0066] Drug solution is injected into the vacuoles through the drug delivery channel;

[0067] Based on the orientation of the fish in the bubble, determine whether the fish needs to be pushed into the second observation channel after entering the first observation channel in order to observe the fish.

[0068] In this embodiment, the liquid outlet channel is used to output liquid, enabling fish to move within the channel or hose; the fish release channel provides a route for releasing the fish after the experiment is completed. The loading module and drug release imaging module use several pumps and switches to move the fish within the hose or channel. The control submodules of the loading module and drug release imaging module regulate the operation of the pumps and switches, achieving automated fish loading. It should be noted that in this embodiment, gas injected through the gas inlet channel divides the bubble, and drug is injected into the bubble through the drug release channel to achieve short-term drug concentration control. The drug release imaging module is used to: if the fish or bubble moves to the junction of the drug release channel and the main channel, inject drug solution into the bubble through the drug release channel. It is understood that in this embodiment, the fish can be observed through either the first observation channel or the second observation channel; fish facing different directions can also be observed through both the first and second observation channels, which can be set according to actual needs by those skilled in the art. This application does not limit the specific locations of the first and second observation channels; the first and second observation channels are used to change the fish's orientation. That is, the drug release imaging module can also be used to determine whether the fish needs to be pushed into the first observation channel after entering the second observation channel, based on the orientation of the fish in the vacuole, in order to observe the fish. It is understood that the control process in the embodiments of this application can be completed by the control module, or by a separate control unit in the loading module and the drug release imaging module, and this application does not impose any specific limitations.

[0069] Optionally, in the in vivo screening system of this embodiment of the invention, the loading module includes: a first pump for injecting air bubbles into the bottom of the fish tank to allow fish to enter the main hose inserted into the fish tank; a second pump connected to the main hose through the first hose; a third pump connected to the main hose through the second hose; a first switch, a second switch, and a third switch are provided on the main hose near the fish tank; a fourth switch is provided on the main hose near the drug release imaging module; a first image acquisition device for acquiring a first image of a first region, the first region being the main hose region between the first hose and the second hose; the loading module is used to: turn on the first pump and the third pump, and turn on the first switch and the third switch to draw the fish into the main hose and push the fish to move in the main hose; if the first image contains fish, turn on the second pump, and turn on the second switch and the fourth switch to push the fish to the drug release imaging module.

[0070] In some possible implementations, refer to Figure 1 As shown, the first and second pumps are injection pumps that move forward, and the third pump is an injection pump that moves backward.

[0071] Optionally, in the in vivo screening system of this embodiment, the drug release imaging module includes: a fourth pump connected to the gas inlet channel via a fifth switch, a sixth switch connected to the liquid outlet channel, a fifth pump connected to the drug release channel, a seventh pump connected to the second observation channel via a seventh switch, a sixth pump connected to the first observation channel via an eighth switch, and a ninth switch connected to the fish release channel; a second image acquisition device for acquiring images of each channel of the microfluidic chip, wherein the second region is the main channel region between the fish loading channel and the liquid outlet channel; the drug release imaging module is used to: if the second region contains a fish and its orientation is the same as a preset orientation, turn on the fourth pump and the sixth pump, and... Turn on the fifth and eighth switches to split the bubble; turn on the fifth pump to inject the drug solution into the bubble; wait for the fish to move to the first observation channel, and observe and analyze the fish using the image processing device to complete the experiment; or, if the second area contains a fish and its orientation is different from the preset orientation, turn on the fourth and sixth pumps, and turn on the fifth and eighth switches to split the bubble; turn on the fifth pump to inject the drug solution into the bubble; wait for the fish to move to the first observation channel, control the sixth pump to push forward and the seventh pump to retract backward, turn on the seventh and eighth switches to move the fish from the first observation channel to the second observation channel; observe and analyze the fish using the image processing device to complete the experiment.

[0072] In some possible implementations, the sixth switch is used to open the water outlet in the channel to allow the fish to move along the hose / channel; the ninth switch is used to release the fish. Specifically, this can be achieved using a second image acquisition device (exemplarily, such as...). Figure 1 The camera 2) determines the specific location of the fish, and then controls the state of each pump and switch; it can also determine the fish's moving speed based on the power of each pump, and then determine the time it takes for the fish to reach each interface, and then control each pump and switch; this application does not limit the specific control method. It is understood that the above embodiments provide the control logic executed when both observation channels can perform image acquisition and recognition.

[0073] In other embodiments, the fish can also be observed and analyzed through one of the observation channels. Specifically, if the second region contains a fish and its orientation is the same as the preset orientation, the fourth and sixth pumps are turned on, and the fifth and eighth switches are turned on to split the bubble; the fifth pump is turned on to inject the drug solution into the bubble; once the fish moves to the first observation channel, the fish is observed and analyzed by the image processing device to complete the experiment; or, if the second region contains a fish and its orientation is different from the preset orientation, the fourth and seventh pumps are turned on, and the fifth and seventh switches are turned on to split the bubble; the fifth pump is turned on to inject the drug solution into the bubble; once the fish moves to the second observation channel, the seventh pump is pushed forward and the sixth pump is pulled back; the seventh and eighth switches are turned on to move the fish from the second observation channel to the first observation channel; the fish is observed and analyzed by the image processing device to complete the experiment.

[0074] Optionally, in the in vivo screening system of this embodiment, the cross-sectional area of ​​the first observation channel near the main channel is the first area, and the cross-sectional area of ​​the first observation channel away from the main channel is the second area, wherein the first area is greater than the second area; the portion of the first observation channel away from the main channel is flat.

[0075] Optionally, in the in vivo screening system of this embodiment, the fish pond is a funnel-shaped container that is wider at the top and narrower at the bottom.

[0076] Next, the in vivo screening method according to an embodiment of the present invention will be described with reference to the accompanying drawings.

[0077] This invention provides an in-vivo screening method. This in-vivo screening method can be applied to a terminal, a server, or software running on a terminal or server. The terminal can be a tablet computer, a laptop computer, a desktop computer, etc., but is not limited to these. The server can be an independent physical server, a server cluster or distributed system composed of multiple physical servers, or a cloud server providing basic cloud computing services such as cloud services, cloud databases, cloud computing, cloud functions, cloud storage, network services, cloud communication, middleware services, domain name services, security services, CDN, and big data and artificial intelligence platforms. The in-vivo screening method of this invention, applied to the above-mentioned in-vivo screening system, mainly includes the following steps:

[0078] Remove the fish from the fish tank and move it through the main hose;

[0079] If a fish enters the drug delivery imaging module, gas is injected to divide the vacuoles containing the fish and to propel the vacuoles through the main channel by the injected gas.

[0080] If the fish reaches the junction of the drug release channel and the main channel, inject the drug solution into the vacuoles;

[0081] Based on the orientation of the fish in the bubble, determine whether the fish needs to be pushed into the second observation channel after entering the first observation channel in order to observe the fish.

[0082] Optionally, in the in vivo screening method of this embodiment, a first pump is connected to the bottom of the fishpond, a second pump is connected to the main hose via a first flexible tube, and a third pump is connected to the main hose via a second flexible tube; a first switch is provided on the main hose near the fishpond, a second switch is provided on the first hose, a third switch is provided on the second hose, and a fourth switch is provided on the main hose near the drug release imaging module; a first image acquisition device is used to acquire a first image of a first region, the first region being the main hose region between the first and second flexible tubes, and the method further includes:

[0083] Turn on the first and third pumps, and turn on the first and third switches to draw the fish into the main hose and push the fish to move in the main hose;

[0084] If the first image contains a fish, turn on the second pump, and turn on the second and fourth switches to push the fish to the drug release imaging module.

[0085] Optionally, in the in vivo screening method of this embodiment, the drug release imaging module includes: a fourth pump connected to the gas inlet channel via a fifth switch, a sixth switch connected to the liquid outlet channel, a fifth pump connected to the drug release channel, a seventh pump connected to the second observation channel via a seventh switch, a sixth pump connected to the first observation channel via an eighth switch, and a ninth switch connected to the fish release channel; a second image acquisition device for acquiring images of each channel of the microfluidic chip, wherein the second region is the main channel region between the fish loading channel and the liquid outlet channel, and the method further includes:

[0086] If the second area contains a fish and its orientation is the same as the preset orientation, turn on the fourth and sixth pumps, and turn on the fifth and eighth switches to split the bubble; turn on the fifth pump to inject the drug solution into the bubble; wait for the fish to move to the first observation channel, and observe and analyze the fish through the image processing device to complete the experiment;

[0087] or,

[0088] If the second area contains a fish and its orientation differs from the preset orientation, turn on the fourth and sixth pumps, and turn on the fifth and eighth switches to split the bubble; turn on the fifth pump to inject the drug solution into the bubble; once the fish moves to the first observation channel, control the sixth pump to push forward and the seventh pump to retract backward, and turn on the seventh and eighth switches to move the fish from the first observation channel to the second observation channel; observe and analyze the fish using an image processing device to complete the experiment.

[0089] The in vivo screening system and screening method provided in this application are described below with reference to a specific embodiment:

[0090] The system is deployed on the XYZ electric triaxial platform and consists of a microfluidic chip, specific silicone tubing, nine electromagnetic clamp valves, seven microfluidic injection pumps, a control circuit board for controlling the electromagnetic clamp valves and microfluidic injection pumps, three CMOS cameras, a fluorescence module (installed with camera 3), and a digital image processing unit. The system schematic is shown below. Figure 1 As shown, it can be divided into a loading module and a drug release and imaging module (i.e., the drug release imaging module mentioned above).

[0091] For example, the operating speed of pumps 1, 2, and 3 can be set to approximately 30 μL / s, and the operating speed of pumps 4, 5, 6, and 7 can all be set to 40 μL / min. Of course, those skilled in the art can adjust these speeds according to actual needs, and this application does not impose any specific limitations.

[0092] like Figure 2 The loading module consists of a conical fish tank, several silicone tubing, a T-junction, camera 1, three sets of microfluidic injection pumps, and several electromagnetic clamp valves. Injection pump 1 connects to the bottom of the fish tank and injects air bubbles to effectively prevent zebrafish fry from sinking. At this time, by opening valves 1 and 3 and closing other valves, injection pump 1 is pushed forward, and injection pump 3 is pulled backward, efficiently drawing liquid and zebrafish fry from the fish tank. When the zebrafish fry appear in the field of view of camera 1 (i.e., the first area mentioned above), the digital image processing unit, equipped with a visual algorithm, responds immediately. The system controls the start and stop of the electromagnetic clamp valves and injection pumps to immediately switch the fluid pathway (opening valves 2, 4, and 6, closing other valves, pushing injection pump 2 forward, and stopping the other injection pumps), driving injection pump 2 to push the liquid segment containing the zebrafish within the field of view of camera 1 to the drug release and imaging module.

[0093] In the drug delivery and imaging module, the microfluidic chip is made of PDMS material (of course, it can also be made of other materials, such as PMMA), as shown in the attached figure. Figure 3 It consists of a zebrafish inlet channel, a gas inlet, a liquid outlet, a drug release channel, observation channel 1, observation channel 2, and a zebrafish release channel. To limit unexpected spontaneous movement of the zebrafish and prevent harm, the channel dimensions may be designed to be similar to or slightly larger than the zebrafish's size. In some embodiments, observation channel 1 / observation channel 2 may be designed as an auxiliary channel. Figure 4The pattern shown in the figure allows for stable fixation of zebrafish and posing them in a sideways posture. Combined with a high-resolution camera and fluorescence module, this perspective enables bright-field or fluorescence optical observation of various organs. Figures 5(a), 5(b), and 5(c) show representative fluorescence and bright-field images of the heart, blood vessels, and liver of Cmlc2:eGFP(d), Kdrl1:eGFP(e), and Apo14:GFP(f) transgenic animals, respectively. Of course, those skilled in the art can design the observation channel in other ways.

[0094] Camera 2's field of view covers all channels of the microfluidic chip. When the loading module delivers the bubble containing the fish between the gas injection channel and the liquid outlet, the digital imaging module sends instructions to the circuit control board, simultaneously transmitting the visual algorithm's calculation of the zebrafish's head and tail orientation. This process is illustrated in the attached diagram. Figure 6 As shown, after the original image undergoes several morphological processing steps including grayscale conversion, median filtering, erosion, and dilation, the zebrafish's eye features become more prominent. Subsequently, edge detection captures these features to obtain the zebrafish's coordinates. The visual algorithm in the loading module works similarly. In the visual algorithm of the drug release and imaging module, the zebrafish is further tracked, and its real-time coordinates are fed back to the grayscale image. By comparing the grayscale values ​​on the left and right sides of the eye contour, it can be determined whether the zebrafish is facing left or right (if the grayscale value is higher on the left side of the eye, it means that most of the zebrafish's body is on the left side of the eye, and the zebrafish's head is facing right, and vice versa).

[0095] When the zebrafish reaches the second area, the control circuit board immediately opens valves 5 and 8, closes other valves, pushes injection pump 4 forward, pulls injection pump 6 backward, closes other pumps, and pushes gas into the injection channel, splitting the bubble. This causes a bubble containing the zebrafish, with a length equal to the distance from the injection channel to the liquid outlet, to be pushed towards observation channel 1. As the bubble passes through the drug release channel, injection pump 5 is pushed forward to inject the drug solution into the bubble. Next, if the zebrafish's tail is facing its direction of movement, all valves are closed and all pumps are stopped, directly fixing the zebrafish in observation channel 1. If the zebrafish's head is facing its direction of movement, after it is pushed into observation channel 1, valves 7 and 8 are opened, other valves are closed, injection pump 6 is pushed forward, injection pump 7 is pulled backward, and other pumps are stopped, immediately transferring and fixing the zebrafish to observation channel 2. At this point, the XYZ electric three-axis platform is used to switch camera 2 to a high-resolution camera 3, which, in conjunction with the fluorescence module, enables multi-organ optical imaging. After observation, the zebrafish is released from the chip by opening valves 8 and 9, closing other valves, and pushing the syringe pump 6 forward (if the zebrafish is being observed in observation channel 1), or by opening valves 7 and 9 and pushing the syringe pump 7 forward (if the zebrafish is being observed in observation channel 2). The above process is shown in the attached diagram. Figure 7 and attached Figure 8 As shown. It is understandable that... Figure 1 The positional relationship of each channel can be adjusted according to actual needs. For example, the positions of the first and second observation channels can be misaligned to achieve automated biological screening. In some embodiments, the cross-sectional area of ​​the gas inlet channel and the liquid outlet channel is less than or equal to the cross-sectional area of ​​the main channel to prevent fish from accidentally entering the gas inlet channel and the liquid outlet channel.

[0096] Figure 9 illustrates the system's ability to generate precisely concentrated drug solutions, as determined by a micro-ultraviolet spectrophotometer. Figure 9(a) shows a conceptual diagram and corresponding photographs of a series of drug release processes, scale bar, 2 mm. In the experiment shown in Figure 9(b), the molar absorbance of methylene blue was measured using a detection wavelength of 664 nm (also used thereafter); five standard solutions of different concentrations (5 μM, 20 μM, 50 μM, 80 μM, and 110 μM) were tested, showing a linear relationship between absorbance and drug concentration within these concentration ranges. The system was then used to dilute the 110 μM solution to 50 μM (error bars represent the standard error (sem) of the mean, n = 5). Figure 9(c) shows the concentration comparison, and Figure 9(d) shows the results of the dilution experiment, displaying the absorbance of the initial and final solutions after 1 minute of diffusion and transfer in the automated microfluidic chip system, as expected. Figure 9(e) shows the absorption spectra of methylene blue solutions ranging from 10 μM to 100 μM generated by the chip. The actual concentration gradient was calculated based on the straight line fitted by the correlation equation between absorbance in Figure 9(e) (as shown in Figure 9(f)).

[0097] As attached Figure 10 As shown, the time required for each step of the system's operation is calculated. This can be used for adjusting individual pumps and switches.

[0098] As shown in Figure 11, the quantitative health assessment of zebrafish after operation using this system was statistically analyzed. The survival rate (Figure 11(a)) and abnormality rate (Figure 11(b)) of zebrafish larvae were observed over the following four days after capture in the automated microfluidic system, two hours later. Five independent experiments (8 larvae / experiment) were used to screen 40 larvae. Error bars represent the standard error (sem) of the mean; one-way ANOVA showed no significant difference in "ns".

[0099] In one embodiment, the automatic loading, transport, drug delivery, and imaging of zebrafish were completed strictly according to the workflow described in the background art. The system was used to verify whether there was a significant difference between bright field and fluorescence field detection. After data analysis, the results are shown in Figure 12.

[0100] Figure 12(a) shows the larval imaging within half of the observation channel of the system's microfluidic chip. Scale bar, 100 μm. Figure 12(b) shows heart rate measurement based on the grayscale value change of the region of interest (ROI) at the center of the ventricle. Scale bar, 100 μm. Figure 12(c) analyzes the motion of the ventricular wall during the cardiac cycle by fitting two-dimensional ellipses on the major and minor axes. Unit time interval, 500 ms. Figures 12(d) and 12(e) show cardiac cycle sequence images of wild-type zebrafish (WT) and transgenic zebrafish (TG) (Cmlc2:eGFP), respectively. Scale bar, 100 μm. Figure 12(f) shows the changes in grayscale value at the center of the ventricle in WT and TG zebrafish. The four comparison relationships on the right are quantitative comparisons of heart rate (HR), fractional area change (FAC), stroke volume (SV), and cardiac output (CO). Statistical results show no significant differences between WT and TG zebrafish.

[0101] In another embodiment, the automated loading, transport, drug delivery, and imaging of zebrafish were completed strictly according to the workflow described in the background art. After testing and analysis, test results were obtained for 10 concentration gradients of sertindole from 10 μM to 100 μM. We analyzed the cardiac physiological activities of zebrafish at different concentrations. As the concentration increased, the cardiotoxicity gradually increased. After the concentration exceeded 20 μM, the cardiac output and ejection fraction decreased significantly, and after the concentration exceeded 80 μM, the heart rate decreased significantly.

[0102] The embodiments of this application can automatically load, transport, and orient zebrafish, which is more efficient than manual labor, as shown in the attached figure. Figure 10 As shown in the illustration. The embodiments of this application can automatically dilute and administer drugs, making it more user-friendly for researchers unfamiliar with zebrafish experiments. Furthermore, the end-to-end automation reduces human intervention, accelerates drug toxicity screening, and further simplifies the drug screening process.

[0103] This invention provides an in vivo screening device, comprising:

[0104] At least one processor;

[0105] At least one memory for storing at least one program;

[0106] When the at least one program is executed by the at least one processor, the at least one processor implements the in vivo screening method.

[0107] Similarly, the content of the above method embodiments is applicable to this device embodiment. The specific functions implemented by this device embodiment are the same as those of the above method embodiments, and the beneficial effects achieved are also the same as those achieved by the above method embodiments.

[0108] This invention also provides a computer-readable storage medium storing a processor-executable program, which, when executed by a processor, performs the in vivo screening method described above.

[0109] Similarly, the content of the above method embodiments is applicable to this storage medium embodiment. The specific functions implemented in this storage medium embodiment are the same as those in the above method embodiments, and the beneficial effects achieved are also the same as those achieved in the above method embodiments.

[0110] In some alternative embodiments, the functions / operations mentioned in the block diagrams may not occur in the order shown in the operation diagrams. For example, depending on the functions / operations involved, two consecutively shown blocks may actually be executed substantially simultaneously, or the blocks may sometimes be executed in reverse order. Furthermore, the embodiments presented and described in the flowcharts of this invention are provided by way of example to provide a more comprehensive understanding of the technology. The disclosed methods are not limited to the operations and logic flows presented herein. Alternative embodiments are contemplated in which the order of various operations is altered and sub-operations described as part of a larger operation are executed independently.

[0111] Furthermore, although the invention has been described in the context of functional modules, it should be understood that, unless otherwise stated, one or more of the functions and / or features may be integrated into a single physical device and / or software module, or one or more functions and / or features may be implemented in a separate physical device or software module. It is also understood that a detailed discussion of the actual implementation of each module is unnecessary for understanding the invention. Rather, given the properties, functions, and internal relationships of the various functional modules in the apparatus disclosed herein, the actual implementation of the module will be understood within the scope of conventional skill of an engineer. Therefore, those skilled in the art can implement the invention as set forth in the claims using ordinary techniques without excessive experimentation. It is also understood that the specific concepts disclosed are merely illustrative and not intended to limit the scope of the invention, which is determined by the full scope of the appended claims and their equivalents.

[0112] If the aforementioned functions are implemented as software functional units and sold or used as independent products, they can be stored in a computer-readable storage medium. Based on this understanding, the technical solution of this invention, or the part that contributes to the prior art, or a part of the technical solution, can be embodied in the form of a software product. This computer software product is stored in a storage medium and includes several programs to cause a computer device (which may be a personal computer, server, or network device, etc.) to execute all or part of the steps of the methods described in the various embodiments of this invention. The aforementioned storage medium includes various media capable of storing program code, such as USB flash drives, portable hard drives, read-only memory (ROM), random access memory (RAM), magnetic disks, or optical disks.

[0113] The logic and / or steps represented in the flowchart or otherwise described herein, for example, can be considered as a sequential list of executable programs for implementing logical functions, and can be embodied in any computer-readable medium for use by, or in conjunction with, a program execution system, apparatus, or device (such as a computer-based system, a processor-included system, or other system that can retrieve and execute a program from or in conjunction with such a program execution system, apparatus, or device). For the purposes of this specification, "computer-readable medium" can mean any means that can contain, store, communicate, propagate, or transmit a program for use by or in conjunction with a program execution system, apparatus, or device.

[0114] More specific examples of computer-readable media (a non-exhaustive list) include: electrical connections (electronic devices) having one or more wires, portable computer disk drives (magnetic devices), random access memory (RAM), read-only memory (ROM), erasable and editable read-only memory (EPROM or flash memory), fiber optic devices, and portable optical disc read-only memory (CDROM). Furthermore, computer-readable media can even be paper or other suitable media on which the program can be printed, since the program can be obtained electronically, for example, by optically scanning the paper or other medium, followed by editing, interpreting, or otherwise processing as necessary, and then stored in computer memory.

[0115] It should be understood that various parts of the present invention can be implemented in hardware, software, firmware, or a combination thereof. In the above embodiments, multiple steps or methods can be implemented in software or firmware stored in memory and executed by a suitable program execution system. For example, if implemented in hardware, as in another embodiment, it can be implemented using any one or a combination of the following techniques known in the art: discrete logic circuits having logic gates for implementing logical functions on data signals, application-specific integrated circuits (ASICs) having suitable combinational logic gates, programmable gate arrays (PGAs), field-programmable gate arrays (FPGAs), etc.

[0116] In the foregoing description of this specification, references to terms such as "one embodiment," "another embodiment," or "some embodiments" indicate that a specific feature, structure, material, or characteristic described in connection with an embodiment or example is included in at least one embodiment or example of the present invention. In this specification, illustrative expressions of the above terms do not necessarily refer to the same embodiment or example. Furthermore, the specific features, structures, materials, or characteristics described may be combined in any suitable manner in one or more embodiments or examples.

[0117] Although embodiments of the invention have been shown and described, those skilled in the art will understand that various changes, modifications, substitutions and alterations can be made to these embodiments without departing from the principles and spirit of the invention, the scope of which is defined by the claims and their equivalents.

[0118] The above is a detailed description of the preferred embodiments of the present invention. However, the present invention is not limited to the embodiments described. Those skilled in the art can make various equivalent modifications or substitutions without departing from the spirit of the present invention. All such equivalent modifications or substitutions are included within the scope defined by the claims of the present invention.

Claims

1. An in vivo screening system, characterized in that, The screening system includes: a loading module and a drug release imaging module; The loading module is used to remove fish from the fishpond and push the fish through the main hose. The drug release imaging module includes a microfluidic chip, which includes a main channel and several channels connected to the main channel. The several channels include a gas inlet channel, a fish loading channel, a liquid outlet channel, a drug release channel, a first observation channel, a second observation channel, and a fish release channel arranged sequentially along the movement direction of the fish. The fish loading channel is connected to the output channel of the loading module to receive fish from the output channel; the gas inlet channel is used to inject gas to divide the vacuoles containing the fish and to propel the vacuoles in the main channel by the injected gas; the drug release imaging module is used for: A drug solution is injected into the vacuoles through a drug release channel; Based on the orientation of the fish in the bubble, determine whether the fish needs to be pushed into the second observation channel after entering the first observation channel in order to observe the fish; The drug release imaging module includes: A fourth pump connected to the gas inlet channel via a fifth switch, a sixth switch connected to the liquid outlet channel, a fifth pump connected to the drug release channel, a seventh pump connected to the second observation channel via a seventh switch, a sixth pump connected to the first observation channel via an eighth switch, and a ninth switch connected to the fish release channel; a second image acquisition device for acquiring images of each channel of the microfluidic chip; the second region is the main channel region between the fish loading channel and the liquid outlet channel; The drug release imaging module is used for: If the second area contains the fish and its orientation is the same as the preset orientation, turn on the fourth and sixth pumps, and turn on the fifth and eighth switches to divide the vacuoles; turn on the fifth pump to inject the drug solution into the vacuoles; wait for the fish to move to the first observation channel, acquire the first image of the fish through the second image acquisition device, and observe and analyze the first image of the fish through the image processing device to complete the experiment; or, If the second region contains the fish and its orientation differs from the preset orientation, the fourth and sixth pumps are activated, as are the fifth and eighth switches, to separate the vacuoles. The fifth pump is activated to inject the drug solution into the vacuoles. Once the fish moves to the first observation channel, the sixth pump is pushed forward, the seventh pump is retracted, and the seventh and eighth switches are activated to move the fish from the first observation channel to the second observation channel. A second image of the fish is acquired using the second image acquisition device. The second image of the fish is then observed and analyzed using the image processing device to complete the experiment.

2. The in vivo screening system according to claim 1, characterized in that, The loading module includes: A first pump is used to inject air bubbles into the bottom of the fishpond to allow fish to enter the main hose inserted into the fishpond. A second pump is connected to the main hose through the first hose, and a third pump is connected to the main hose through the second hose. A first switch is provided on the main hose near the fishpond, a second switch is provided on the first hose, a third switch is provided on the second hose, and a fourth switch is provided on the main hose near the drug release imaging module. A first image acquisition device is used to acquire a first image of a first region, which is the main hose region between the first hose and the second hose. The loading module is used for: Turn on the first pump and the third pump, and turn on the first switch and the third switch to draw the fish into the main hose and push the fish to move in the main hose; if the first image contains the fish, turn on the second pump, and turn on the second switch and the fourth switch to push the fish to move to the drug release imaging module.

3. The in vivo screening system according to claim 1, characterized in that, The cross-sectional area of ​​the first observation channel near the main channel is the first area, and the cross-sectional area of ​​the first observation channel away from the main channel is the second area. The first area is larger than the second area. The portion of the first observation channel away from the main channel is flat.

4. The in vivo screening system according to claim 1, characterized in that, The fishpond is a funnel-shaped container that is wider at the top and narrower at the bottom.

5. An in vivo screening method, characterized in that, Applied to the in vivo screening system as described in claim 1, the method comprises: Remove the fish from the fish tank and move it through the main hose; If the fish enters the drug release imaging module, gas is injected to divide the vacuoles containing the fish and to propel the vacuoles in the main channel by the injected gas. If the fish reaches the connection between the drug release channel and the main channel, the drug solution is injected into the vacuoles; Based on the orientation of the fish in the bubble, determine whether the fish needs to be pushed into the second observation channel after entering the first observation channel in order to observe the fish; The drug release imaging module includes: a fourth pump connected to the gas inlet channel via a fifth switch, a sixth switch connected to the liquid outlet channel, a fifth pump connected to the drug release channel, a seventh pump connected to the second observation channel via a seventh switch, a sixth pump connected to the first observation channel via an eighth switch, and a ninth switch connected to the fish release channel; a second image acquisition device for acquiring images of each channel of the microfluidic chip, wherein the second region is the main channel region between the fish loading channel and the liquid outlet channel; the method further includes: If the second area contains the fish and its orientation is the same as the preset orientation, turn on the fourth and sixth pumps, and turn on the fifth and eighth switches to divide the vacuoles; turn on the fifth pump to inject the drug solution into the vacuoles; wait for the fish to move to the first observation channel, acquire the first image of the fish through the second image acquisition device, and observe and analyze the first image of the fish through the image processing device to complete the experiment; or, If the second region contains the fish and its orientation differs from the preset orientation, the fourth and sixth pumps are activated, as are the fifth and eighth switches, to separate the vacuoles. The fifth pump is activated to inject the drug solution into the vacuoles. Once the fish moves to the first observation channel, the sixth pump is pushed forward, the seventh pump is retracted, and the seventh and eighth switches are activated to move the fish from the first observation channel to the second observation channel. A second image of the fish is acquired using the second image acquisition device. The second image of the fish is then observed and analyzed using the image processing device to complete the experiment.

6. The in vivo screening method according to claim 5, characterized in that, A first pump is connected to the bottom of the fishpond; a second pump is connected to the main hose via a first flexible tube; a third pump is connected to the main hose via a second flexible tube; a first switch is located on the main hose near the fishpond; a second switch is located on the first flexible tube; a third switch is located on the second flexible tube; and a fourth switch is located on the main hose near the drug release imaging module; a first image acquisition device is used to acquire a first image of a first region, the first region being the main hose region between the first flexible tube and the second flexible tube; the method further includes: Turn on the first pump and the third pump, turn on the first switch and the third switch to draw the fish into the main hose and push the fish to move in the main hose; If the first image contains a fish, turn on the second pump, the second switch, and the fourth switch to push the fish to the drug release imaging module.

7. An in vivo screening device, characterized in that, include: At least one processor; At least one memory for storing at least one program; When the at least one program is executed by the at least one processor, the at least one processor implements the in vivo screening method as described in any one of claims 5-6.

8. A computer-readable storage medium storing a processor-executable program, characterized in that, The processor-executable program, when executed by the processor, is used to implement the in vivo screening method as described in any one of claims 5-6.

Images